Cloning and sequencing of the yeast gene for dolichol phosphate mannose synthase, an essential protein.
Cloning and sequencing of the yeast gene for dolichol phosphate mannose synthase, an essential protein.
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发表时间：
1988-11
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通讯作者：
P. Orlean;C. Albright;P. Robbins
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作者：
P. Orlean;C. Albright;P. Robbins
Dolichol磷酸甘露糖（Dol-P-Man）合酶（EC 2.4.1.83）催化Dol-P和GDP-Man形成Dol-P-Man。通过筛选酵母基因组DNA文库中过表达Dol-P-Man合酶活性的菌落来分离酵母Dol-P-Man合酶（DPM 1）的结构基因。该方法依赖于在裂解的酵母菌落中筛选Dol-P-Man合酶活性的方法，并使用在菌落裂解物中具有非常低的Dol-P-Man合酶活性的酵母突变体。使用该技术分离的转化体表达比野生型菌株高9-14倍的Dol-P-Man合酶活性，并且赋予这种过量生产的所有七种质粒在其酵母基因组DNA插入物中具有共同区域。DPM 1是酵母Dol-P-Man合酶的结构基因，因为携带该基因的大肠杆菌转化体在体外表达Dol-P-Man合酶活性。DPM 1基因的DNA测序揭示了801个碱基的开放阅读框架。预测的蛋白质的30-kDa大小与纯化的酵母酶的大小非常一致（Haselbeck，A.，和坦纳，W.等人（1982）Proc. Acad. Sci.联合S. A. 79，1520-1524）。预测的氨基酸序列的分析表明，该蛋白质在其COOH末端具有25个氨基酸的潜在跨膜结构域。该蛋白的NH 2末端虽然不疏水，但符合酵母信号序列的现有标准，但没有信号肽酶切割的位点。如果NH 2末端是功能性信号序列，则预测该蛋白质朝向内质网的内腔，其中NH 2和COOH末端都充当膜锚。如果没有信号序列，则预测酶面向细胞质并且仅通过其COOH末端锚定。DPM 1基因对于酵母的生存能力至关重要，因为该基因的破坏是致命的。我们怀疑Dol-P-Man合成酶不是一种必需蛋白，因为它在N-糖基化中的作用，因为影响脂联寡糖合成后期步骤的其他基因突变不影响细胞生长。相反，DPM 1可能是一个必需的基因，因为它的产物是酵母中O-糖基化所必需的，或者因为Dol-P-Man合酶在一些未知的途径中是必需的。
Dolichol phosphate mannose (Dol-P-Man) synthase (EC 2.4.1.83) catalyzes the formation of Dol-P-Man from Dol-P and GDP-Man. The structural gene for yeast Dol-P-Man synthase (DPM1) was isolated by screening a yeast genomic DNA library for colonies that overexpressed Dol-P-Man synthase activity. This approach relied on a method to screen for Dol-P-Man synthase activity in lysed yeast colonies and used a yeast mutant with very low Dol-P-Man synthase activity in colony lysates. Transformants isolated using this technique expressed Dol-P-Man synthase activity 9-14-fold higher than that of a wild type strain, and all seven plasmids conferring this overproduction had a common region in their yeast genomic DNA insert. DPM1 is the structural gene for yeast Dol-P-Man synthase since Escherichia coli transformants harboring this gene express Dol-P-Man synthase activity in vitro. DNA sequencing of the DPM1 gene revealed an open reading frame of 801 bases. The 30-kDa size of the predicted protein is in excellent agreement with the size of the purified yeast enzyme (Haselbeck, A., and Tanner, W. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 1520-1524). Analysis of the predicted amino acid sequence reveals the protein has a potential membrane spanning domain of 25 amino acids at its COOH terminus. The protein's NH2 terminus, though not hydrophobic, meets existing criteria for yeast signal sequences, but there is no site for cleavage by signal peptidase. If the NH2 terminus is a functional signal sequence, the protein is predicted to be oriented toward the lumen of the endoplasmic reticulum with both NH2 and COOH termini serving as membrane anchors. If there is no signal sequence, the enzyme is predicted to face the cytoplasm and be anchored only by its COOH terminus. The DPM1 gene is essential for viability in yeast since disruption of the gene is lethal. We suspect Dol-P-Man synthase is not an essential protein due to its role in N-glycosylation since mutations in other genes that affect the late steps in lipid-linked oligosaccharide synthesis do not affect cell growth. Instead, DPM1 may be an essential gene because its product is required for O-glycosylation in yeast or because Dol-P-Man synthase is needed in some unidentified pathway.